PDA: Pooled DNA analyzer

Background  

Association mapping using abundant single nucleotide polymorphisms is a powerful tool for identifying disease susceptibility genes for complex traits and exploring possible genetic diversity. Genotyping large numbers of SNPs individually is performed routinely but is cost prohibitive for large-scale genetic studies. DNA pooling is a reliable and cost-saving alternative genotyping method. However, no software has been developed for complete pooled-DNA analyses, including data standardization, allele frequency estimation, and single/multipoint DNA pooling association tests. This motivated the development of the software, 'PDA' (Pooled DNA Analyzer), to analyze pooled DNA data.     

HtrA1 inhibits mineral deposition by osteoblasts: requirement for the protease and PDZ domains

HtrA1 is a secreted multidomain protein with serine protease activity. In light of increasing evidence implicating this protein in the regulation of skeletal development and pathology, we investigated the role of HtrA1 in osteoblast mineralization and identified domains essential for this activity. We demonstrate increased HtrA1 expression in differentiating 2T3 osteoblasts prior to the appearance of mineralization. HtrA1 is subsequently down-regulated in fully mineralized cultures. The functional role of HtrA1 in matrix calcification was investigated using three complementary approaches. First, we transfected a full-length HtrA1 expression plasmid into 2T3 cells and showed that overexpression of HtrA1 delayed mineralization, reduced expression of Cbfa1 and collagen type I mRNA, and prevented BMP-2-induced mineralization. Second, knocking down HtrA1 expression using short interfering RNA induced mineral deposition by 2T3 cells. Third, by expressing a series of recombinant HtrA1 proteins, we demonstrated that the protease domain and the PDZ domain are essential for the inhibitory effect of HtrA1 on osteoblast mineralization. Finally, we tested whether HtrA1 cleaves specific matrix proteins that are known to regulate osteoblast differentiation, mineralization, and/or BMP-2 activity. Full-length recombinant HtrA1 cleaved recombinant decorin, fibronectin, and matrix Gla protein. Both the protease domain and the PDZ domain were necessary for the cleavage of matrix Gla protein, whereas the PDZ domain was not required for the cleavage of decorin or fibronectin. Type I collagen was not cleaved by recombinant HtrA1. These results suggest that HtrA1 may regulate matrix calcification via the inhibition of BMP-2 signaling, modulating osteoblast gene expression, and/or via the degradation of specific matrix proteins.     

A large-scale survey of genetic copy number variations among Han Chinese residing in Taiwan

Background

Winter migration of immature brown trout (Salmo trutta) into freshwater rivers has been hypothesized to result from physiologically stressful combinations of high salinity and low temperature in the sea.

Results

We sampled brown trout from two Danish populations entering different saline conditions and quantified expression of the hsp70 and Na/K-ATPases α 1b genes following acclimation to freshwater and full-strength seawater at 2°C and 10°C. An interaction effect of low temperature and high salinity on expression of both hsp70 and Na/K-ATPase α 1b was found in trout from the river entering high saline conditions, while a temperature independent up-regulation of both genes in full-strength seawater was found for trout entering marine conditions with lower salinities.

Conclusion

Overall our results support the hypothesis that physiologically stressful conditions in the sea drive sea-run brown trout into freshwater rivers in winter. However, our results also demonstrate intra-specific differences in expression of important stress and osmoregulative genes most likely reflecting adaptive differences between trout populations on a regional scale, thus strongly suggesting local adaptations driven by the local marine environment.     

Evaluation of approaches for measuring taxonomic completeness of lake profundal macroinvertebrate assemblages

1. Benthic macroinvertebrates (MI) are commonly used to assess freshwater ecosystems with the reference condition approach. Such assessments necessitate control for natural community variation, either by categorical typologies or by predictive models that have been widely and successfully developed for running water biota but not previously for lake profundal invertebrates. 2. We evaluated four modelling techniques [multivariate regression tree (MRT), limiting environmental differences, nonparametric multiplicative regression (NPMR) and River Invertebrate Prediction And Classification System (RIVPACS) and the operative Finnish lake typology for assessing taxonomic completeness (observed‐to‐expected number of taxa, O/E) of profundal MI assemblages. We used data from 74 and 33 minimally disturbed reference lake basins for calibration and validation of the approaches, respectively, and 72 test basins subject to various anthropogenic pressures to evaluate sensitivity to detect impact. Either all predicted taxa (threshold probability of capture P_t = 0+) or only those predicted to be captured with ≥0.25 probability were used to calculate O/E. 3. With P_t = 0.25, all four modelling approaches were accurate (mean O/E = 0.966–1.053) but imprecise (SD of O/E = 0.279–0.304) in predicting the fauna actually observed in validation sites. All models were subtly more precise than a null model (mean 1.038, SD 0.343) or the typology (1.046, 0.327). The taxon‐specific NPMR model was slightly more precise than the other three models based on site groupings. 4. The O/E values correlated relatively weakly (r = 0.55–0.86) among the approaches, which thus produced contrasting lake‐specific assessments, despite their seemingly comparable performances. Indeed, typology, suggesting that MI assemblages were impaired in 56% of test sites, was more sensitive than the other approaches (26–46%) as an indicator of human‐induced deterioration. However, this greater ostensible sensitivity seemed to be biased, as lake morphometry, a main driver of natural community variation, remained uncontrolled by the typology. 5. Generally, our exercise illustrates the inconclusiveness of the common validation criteria for the assessment methods. The apparent poor predictability of the profundal fauna, irrespective of the method, may partly stem from large observation error, which could be alleviated by more intensive sampling. However, instead of an O/E‐taxa index, some other metric encompassing quantitative aspects might be preferable for assessing these species‐poor communities.     

Genomic and bioinformatics analysis of HAdV-4, a human adenovirus causing acute respiratory disease: implications for gene therapy and vaccine vector development

Human adenovirus serotype 4 (HAdV-4) is a reemerging viral pathogenic agent implicated in epidemic outbreaks of acute respiratory disease (ARD). This report presents a genomic and bioinformatics analysis of the prototype 35,990-nucleotide genome (GenBank accession no. AY594253). Intriguingly, the genome analysis suggests a closer phylogenetic relationship with the chimpanzee adenoviruses (simian adenoviruses) rather than with other human adenoviruses, suggesting a recent origin of HAdV-4, and therefore species E, through a zoonotic event from chimpanzees to humans. Bioinformatics analysis also suggests a pre-zoonotic recombination event, as well, between species B-like and species C-like simian adenoviruses. These observations may have implications for the current interest in using chimpanzee adenoviruses in the development of vectors for human gene therapy and for DNA-based vaccines. Also, the reemergence, surveillance, and treatment of HAdV-4 as an ARD pathogen is an opportunity to demonstrate the use of genome determination as a tool for viral infectious disease characterization and epidemic outbreak surveillance: for example, rapid and accurate low-pass sequencing and analysis of the genome. In particular, this approach allows the rapid identification and development of unique probes for the differentiation of family, species, serotype, and strain (e.g., pathogen genome signatures) for monitoring epidemic outbreaks of ARD.     

Reproductive effort of Mastocarpus papillatus (Rhodophyta) along the California coast1

Species with sexual and asexual life cycles may exhibit intraspecific differences in reproductive effort. The spatial separation of sexual and asexual lineages, called geographic parthenogenesis, is common in plants, animals, and algae. Mastocarpus papillatus is a well‐documented case of geographic parthenogenesis in which sexuals dominate southern populations, asexuals dominate northern popula‐tions, whereas mixed populations occur throughout central California. We quantified abundances and reproductive effort of sexual and asexual fronds and tetrasporophytes at eight sites in California to test the hypotheses that (1) reduced sexual reproduction at higher latitudes and tidal heights explains the observed geographic parthenogenesis and (2) reproductive effort (spore production per blade area) declines with increasing latitude. Abundances of all phases varied site‐specifically. However, there was no geographic pattern of reproductive effort of fronds. Reproductive effort of fronds was greater in 2006 than in 2007 and correlated with sea surface temperatures. Sexual fronds exhibited greater reproductive effort than did asexual fronds and sexual reproductive effort was also inversely correlated with local upwelling index. Tetrasporophytes showed greater repro‐ductive effort in northern sites, but total supply of tetraspores per m² was greatest in the middle of the sampling range where crusts were more abundant. There was no decline of reproductive effort at higher latitudes. Geographic patterns of fecundity of life stages do not explain geographic parthe‐nogenesis in M. papillatus. Site‐specific differences in viability among spores or established thalli of different life cycles may explain their respective geographic distributions, as the sexual and asexual life cycles responded differently to environmental variations.     

Application of harmonic radar technology to monitor tree snail dispersal

Abstract. Planned conservation efforts for tree snails of the endangered genus Achatinella, endemic to the island of O'ahu, Hawai'i, will include translocations among the remaining wild and captive‐bred populations. In order to establish optimal levels of artificial migration among neighboring groups of snails within fragmented populations, efforts to determine natural dispersal rates through direct observation were initiated. Capture–mark–recapture (CMR) efforts have proved inadequate for obtaining the requisite dispersal estimates, due to low recapture probabilities. In addition, snail dispersal beyond the boundaries of a finite CMR study site was indistinguishable from mortality. In the preliminary study reported here, both the low recapture probability and dispersal detection problems of past CMR efforts were addressed by using harmonic radar tracking. This approach yielded rough dispersal estimates that were unattainable using CMR alone by providing 100% recapture rates even beyond the normal survey area boundaries. Extensive snail movements within clusters of connected trees were frequently observed after tracking for merely a few hours, although movements between unconnected trees were rare and recorded only after monthly survey intervals. Just 11 out of 40 tracked snails made between‐tree movements (average distance of 4.94±1.52 m) during the entire 7‐month study, and provided the only data utilizable for inferring gene flow in and out of subpopulations. Meteorological data loggers were deployed when tracking began to look for an association between such snail movement and weather fluctuations. The resultant data indicate that increases in both wind gusts and humidity facilitate dispersal (R²=0.77, p‐value <0.001), and that passive wind dispersal alone may be responsible for many snail movements (R²=0.59, p‐value=0.0014). Despite having provided coarse estimates of short‐term dispersal and corresponding wind influences, the limitations of the radar method can be substantial.     

Cation-Exchange resin-catalyzed addition of methanol to benzoylated 1,5-anhydro-2-deoxy-d-hex-1-enitols

1,5-Anhydro-3,4,6-tri-O-benzoyl-2-deoxy-d-arabino-hex-1-enitol (1) was boiled under reflux with methanol and AG 50W-X8 cation-exchange resin. A two-product mixture of glycosides (2 and 3) was obtained in 38% yield, together with 19% of unreacted material. 1,5-Anhydro-3,6-di-O-benzoyl-2-deoxy-d-arabino-hex-1-enitol (7) was prepared from 1,5-anhydro-2-deoxy-d-arabino-hex-1-enitol by selective benzoylation, from which the corresponding 4-methanesulfonate 8 was obtained. Treatment of 8 with sodium benzoate in hexamethylphosphoric triamide for 72 h at 100° afforded 1,5-anhydro-3,4,6-tri-O-benzoyl-2-deoxy-d-lyxo-hex-1-enitol (9) in 52% yield. An unknown byproduct (B), tentatively shown to be a tri-O-benzoyl-d-hex-2-enopyranose analog, was also isolated in 14% yield. The 270-MHz n.m.r. spectrum of B was analyzed in terms of its J_1,3, J_2,4, and J_4,5 coupling constants in relation to the various configurational and conformational possibilities for hex-2-enopyranoses, and was identified as 1,4,6-tri-O-benzoyl-2,3-dideoxy-α-d-threo-hex-2-enopyranose having the ^oH₅ conformation. The analysis presented should also be applicable to pent-2-enopyranose systems. When 9 was treated with methanol in the presence of AG 50W-X8 cation-exchange resin, a mixture of glycosides 4 and 5 was obtained in 47% yield. The low yields were attributed to methanolysis of the benzoyl groups during the reaction.     

The spatial scale of local adaptation in a stochastic environment

The distribution of phenotypes in space will be a compromise between adaptive plasticity and local adaptation increasing the fit of phenotypes to local conditions and gene flow reducing that fit. Theoretical models on the evolution of quantitative characters on spatially explicit landscapes have only considered scenarios where optimum trait values change as deterministic functions of space. Here, these models are extended to include stochastic spatially autocorrelated aspects to the environment, and consequently the optimal phenotype. Under these conditions, the regression of phenotype on the environmental variable becomes steeper as the spatial scale on which populations are sampled becomes larger. Under certain deterministic models – such as linear clines – the regression is constant. The way in which the regression changes with spatial scale is informative about the degree of phenotypic plasticity, the relative scale of effective gene flow and the environmental dependency of selection. Connections to temporal models are discussed.